Chemokines are small cytokines having similar structures. There are two types of chemokines: those of the CXC type and those of the CC type, in which four conserved cysteines are linked by disulfide bonds. In the CXC type, the first two cysteines are separated by one amino acid, whereas in the CC type they are not. The two types of chemokines are well known, and described, for example, by Baggioloini et al., 1997.
Interleukin-8 (IL-8) is a representative, well-studied chemokine that is produced by a variety of cell types involved in inflammatory settings. It is known to act on neutrophilic polymorphonuclear leukocytes (neutrophils), promoting their migration to the site of inflammation and causing their subsequent activation (Bacon, K. B. and Schall, T. J. (1996) Int. Arch. Allergy Immunol. 109, 97-109; Baggiolini, M. and Clark-Lewis, I., (1992) FEBS Lett. 307, 97-101). Two receptor subtypes (A and B, also called CXC R1 and CXC R2) are currently known to mediate IL-8 action in cells (Holmes, W. E. et al. (1991) Science 253, 1278-1280; Murphy, P. M. and Tiffany, H. L. (1991) Science 253, 1280-1283). In contrast with receptors for other interleukins, these molecules belong to the large family of seven-transmembrane G protein-coupled receptors.
The amount of IL-8 (and other chemokines) is elevated in a variety of morbid conditions, including lung diseases such as cystic fibrosis and adult respiratory distress syndrome, atherosclerosis, psoriasis, rheumatoid arthritis and reperfusion injury (Strieter et al., (1994) J. Lab. Clin. Med. 123, 183-197). The causal role of IL-8 in the onset of inflammation has been demonstrated in animal models (Sekido N. et al., (1993) Nature 365, 654-657; Leonard, E. J. et al. (1991) J. Invest. Dermatol. 96, 690-694).
This body of evidence has led to efforts to discover antagonists of IL-8 binding to its specific cell surface receptors. Such antagonists are candidates as therapeutic agents for IL-8-dependent disorders. For example, IL-8 labelled with .sup.125 I has been used in assays for binding to CHO membranes to determine IL-8 antagonists, as described in WO 9625157. However, .sup.125 I is a dangerous reagent, posing significant handling and disposal problems. In addition, radioactively iodinated reagents decay when stored for long periods, and cannot be used thereafter. Also, enzymatic processes for radioactively iodinating peptides can, under certain circumstances, cause a loss in binding activity.
Lanthanides are fluorescent labels that have been used primarily in clinical diagnostic kits in standard immunoassay formats; i.e., they have been used to label antibodies for use in such kits. For example, they are conventionally used in time-resolved fluorescent assays for clinical screening. Multiple lanthanide labels (Europium, Samarium, Terbium, and Dysprosmium) can be used in the same assay, and distinguished by their differing emission wavelength/decay time fluorescent profiles. Thus, different analytes can be determined in the same assay using these different labels.
The fluorescent properties of lanthanide probes make them particularly useful in biological assays (Soini, E. and Loevgren, T. (1987) CRC Crit. Rev. Anal. Chem. 18, 105-153). Specifically, the extended fluorescence lifetime of lanthanides allows measurement of signal to be made after the decay of shorter-lived background fluorescence originating from either the biological sample or the plastic support. For example, Eu-labelled anti-phosphotyrosine has been used in a sensitive protein tyrosine kinase assay (Braunwalder et al., (1996) Anal. Biochem. 238, 159-164). Also, Takeuchi, T., et al., ((1995) Anal. Chem. 67, 2655-8) have described a lanthanide-labelled benzodiazepine used in receptor binding assays.
To find lead compounds for drug discovery programs, large numbers of compounds are often screened for their activity as enzyme inhibitors or receptor agonists/antagonists. Large libraries of compounds are needed for such screening. As a result of developments in this field, it is now possible to simultaneously produce combinatorial libraries containing hundreds of thousands of small molecules for screening. With the availability of such libraries, however, has come a need for large-scale, rapid screening methods.
Lanthanide reagents would appear to be potentially useful in high throughput screening of such libraries to measure the binding of chemokines to their receptors. Conjugation of lanthanide chelates, and other detectable labels, to chemokines, however, can, and has, resulted in substantial loss of binding affinity.
In an attempt to obtain a fluorescently labelled IL-8, Alouani, S., et al. (1995) Eur. J. Biochem. 227, 328-334 specifically labelled the amino terminus of IL-8 by first oxidizing the serine at amino acid position no. 1 under mild periodate oxidation conditions and reacting the resultant glyoxylyl with an amino-oxy derivative of fluorescein. The resultant fluorescein-labelled IL-8 had a 10-fold lower affinity than [.sup.125 I]IL-8 for IL-8 receptors. Thus, binding of IL-8 was substantially lost with fluorescein isothiocyanate labeling.
Similarly, modification of a cysteine at position 25 of IL-8 by addition of a fluorescent nitrobenzoxadiazol iodoacetamide (NBD) was found to lower affinity of the fluorescently labelled IL-8 by two orders of magnitude. (Lusti-Narasimhan et al. (1996) J. Biol. Chem. 271, 3148-3153).
IL-8 can be prepared containing lanthanide fluorescent labels conjugated to IL-8 at available lysine groups. This method, however, can modify lysine residues that are critical to the receptor-ligand interaction. Thus, while the resulting reagent may be useful for a competitive immunoassay for determining the amount of IL-8 in a sample, it is less acceptable for use in a competitive assay to measure receptor/ligand binding.
It is therefore an object of the invention to label a polypeptide having the binding affinity of a CC or CXC chemokine without substantially decreasing that affinity.
It is also an object of the invention to obtain a labelled reagent that avoids the handling and disposal problems associated with .sup.125 I.
It is also an object of the invention to provide an assay that is easily adaptable for high throughput screening of potential CC and CXC chemokine receptor ligands.
It is another object of the invention to provide a labelled reagent that can be stored for considerable periods of time without loss of activity.